Objective PARKIN is an E3 ubiquitin ligase that regulates mitochondrial quality control through a process called mitophagy. Recent human and rodent studies suggest that loss of hepatic mitophagy may occur during the pathogenesis of obesity-associated fatty liver and contribute to changes in mitochondrial metabolism associated with this disease. Whole-body Prkn knockout mice are paradoxically protected against diet-induced hepatic steatosis; however, liver-specific effects of Prkn deficiency cannot be discerned in this model due to pleotropic effects of germline Prkn deletion on energy balance and subsequent protection against diet-induced obesity. We therefore generated the first liver-specific Prkn knockout mouse strain (LKO) to directly address the role of hepatic Prkn . Methods Littermate control (WT) and LKO mice were fed regular chow (RC) or high-fat diet (HFD) and changes in body weight and composition were measured over time. Liver mitochondrial content was assessed using multiple, complementary techniques, and mitochondrial respiratory capacity was assessed using Oroboros O 2 K platform. Liver fat was measured biochemically and assessed histologically, while global changes in hepatic gene expression were measured by RNA-seq. Whole-body and tissue-specific insulin resistance were assessed by hyperinsulinemic-euglycemic clamp with isotopic tracers. Results Liver-specific deletion of Prkn had no effect on body weight or adiposity during RC or HFD feeding; however, hepatic steatosis was increased by 45% in HFD-fed LKO compared with WT mice (P < 0.05). While there were no differences in mitochondrial content between genotypes on either diet, mitochondrial respiratory capacity and efficiency in the liver were significantly reduced in LKO mice. Gene enrichment analyses from liver RNA-seq results suggested significant changes in pathways related to lipid metabolism and fibrosis in HFD-fed Prkn knockout mice. Finally, whole-body insulin sensitivity was reduced by 35% in HFD-fed LKO mice (P < 0.05), which was primarily due to increased hepatic insulin resistance (60% of whole-body effect; P = 0.11). Conclusions These data demonstrate that PARKIN contributes to mitochondrial homeostasis in the liver and plays a protective role against the pathogenesis of hepatic steatosis and insulin resistance.

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Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies, including steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD. Mitophagy is a mitochondrial quality control mechanism that allows for selective removal of damaged mitochondria from the cell. Whether changes in mitophagy occur during and contribute to NAFLD is unknown. PARKIN is a ubiquitin E3 ligase that regulates mitophagy by generating a ubiquitin signaling motif on the outer membrane of damaged mitochondria, marking them for degradation. We undertook the studies described to determine effects of genetic inhibition of mitophagy via conditional deletion of PARKIN in liver on progression of NAFLD. Twelve-week old male liver-specific PARKIN knockout (LKO) and wild type (WT) mice were fed a diet previously demonstrated to induce NAFLD that consisted of high-fat (45%), cholesterol (0.15%) and high-fructose corn syrup drinking water (42g/L) for 8 weeks. There was no difference in body weight or composition between groups. Plasma ALT and AST levels were increased 2-fold (p=0.18) and 1.7-fold (p&lt;0.05), respectively, in LKO compared with WT mice, while plasma cholesterol and insulin levels were not different and plasma fatty acid levels were reduced by 27% (p&lt;0.05). NAFLD activity score was increased 2-fold (p&lt;0.05) in LKO mice reflecting an increase in steatosis, ballooning and inflammation. Gene expression markers of NAFLD progression including markers of inflammation (Ccl2, Cd68, Il-1b, ll-6) and fibrosis (Col1a1, Col3a1) were significantly increased in LKO mice. Consistent with these mouse data, we found PARKIN protein expression to be reduced in liver biopsies from patients with NASH compared with healthy controls. These data suggest that loss of PARKIN-mediated mitophagy may contribute to the progression of NAFLD. Disclosure R. Undamatla: None. L.R. Edmunds: None. B. Xie: None. A. Mills: None. I.J. Sipula: None. S.P. Monga: None. M.J. Jurczak: None. Funding National Institutes of Health (DK114012)

High salt diet is associated with increased risk of adverse cardiovascular events due to its effects on blood pressure, vascular stiffening, and fibrosis. The kidney maintains Na+ homeostasis in times of dietary excess by excreting it in urine. Additionally, the brain’s thirst centers sense excess Na+ and increase water intake. Recent work showed mice given high Na+ loads, provided in chow and water, have changes in glucocorticoid, mineralocorticoid, and urea production that lead to increased free water absorption, conservation of fluid with less water intake, and a switch to a catabolic state. However, mice given a salt load were not followed beyond a few weeks of treatment nor were the same results seen when free access to water was given. Therefore, we were interested in the metabolic changes that occur with long‐term dietary Na+ manipulation with free access to water. Mice were fed an 8% NaCl diet (HSD) beginning at~10 weeks of age and given free access to water. After three months, the HSD mice weighed significantly less than the controls (30.1±2.4g vs 33.3±2.8g), and this trend continued through 16 months of HSD treatment when the HSD animals weighed ~10g less than the controls (33.8±1.7g vs 44.8±7.4g). At 12 months, the HSD group consumed more food (19.9±1.0g vs 11.7±0.5g), therefore the weight loss cannot be accounted for through consumption. The HSD cohort also consumed more water (14.5±3.5mL vs 2.0±0.4mL) and produced more urine (16.4±2.7mL vs 4.0±0.2mL) than their control counterparts. At both 8 months and 16 months, the blood urea nitrogen levels in the HSD treated animals were significantly lower, suggesting these animals did not have increased urea production. Additionally, the HSD mice had 12.8% body fat at 8 months treatment as compared to 25.0% body fat in age‐matched controls. However, lean mass by weight was not significantly different between the HSD and control animals (27.9±0.4g vs 28.7±0.4g), suggesting there was no muscle wasting due to salt consumption. To address the physiological mechanism by which high salt intake reduced body fat and weight, mice were studied in metabolic cages to measure the major determinants of energy balance after just 5 weeks HSD, prior to the divergence in body weight. There was no difference in feeding between groups, however water intake was significantly elevated in the HSD animals. Total activity also did not differ between the HSD and control animals. Energy expenditure was significantly increased in HSD mice during the light cycle and there was a strong trend towards increased 24 hour energy expenditure (P=0.09). Finally, the respiratory quotient was significantly reduced in the HSD cohort during both the light and dark cycles, demonstrating increased whole‐body fat oxidation. Taken together, these data demonstrate that animals given an increased salt load have a higher metabolic demand and increased fatty acid oxidation, which were associated with reduced body weight and adiposity. These results suggest that the effects of sodium extend beyond blood press...